The challenge of linking intergenic mutations to target genes has limited molecular understanding of human diseases. Here we show that H3K27ac HiChIP generates high-resolution contact maps of active enhancers and target genes in rare primary human T cell subtypes and coronary artery smooth muscle cells. Differentiation of naive T cells into T helper 17 cells or regulatory T cells creates subtype-specific enhancer–promoter interactions, specifically at regions of shared DNA accessibility. These data provide a principled means of assigning molecular functions to autoimmune and cardiovascular disease risk variants, linking hundreds of noncoding variants to putative gene targets. Target genes identified with HiChIP are further supported by CRISPR interference and activation at linked enhancers, by the presence of expression quantitative trait loci, and by allele-specific enhancer loops in patient-derived primary cells. The majority of disease-associated enhancers contact genes beyond the nearest gene in the linear genome, leading to a fourfold increase in the number of potential target genes for autoimmune and cardiovascular diseases.

CRISPR-Cas genome editing creates targeted DNA double-strand breaks (DSBs) that are processed by cellular repair pathways, including the incorporation of exogenous DNA via single-strand template repair (SSTR). To determine the genetic basis of SSTR in human cells, we developed a coupled inhibition-cutting system capable of interrogating multiple editing outcomes in the context of thousands of individual gene knockdowns. We found that human Cas9-induced SSTR requires the Fanconi anemia (FA) pathway, which is normally implicated in interstrand cross-link repair. The FA pathway does not directly impact error-prone, non-homologous end joining, but instead diverts repair toward SSTR. Furthermore, FANCD2 protein localizes to Cas9-induced DSBs, indicating a direct role in regulating genome editing. Since FA is itself a genetic disease, these data imply that patient genotype and/or transcriptome may impact the effectiveness of gene editing treatments and that treatments biased toward FA repair pathways could have therapeutic value.

21Genome editing using nucleases such as CRISPR-Cas induces programmable DNA damage at a 22 target genomic site but can also affect off-target sites. Here, we develop a powerful, sensitive assay 23 for the unbiased identification of off-target sites that we term DISCOVER-Seq. This approach 24 takes advantage of the recruitment of endogenous DNA repair factors for genome-wide 25 identification of Cas-induced double-strand breaks. One such factor, MRE11, is recruited precisely 26 to double-strand breaks, enabling molecular characterization of nuclease cut sites with single-base 27 resolution. DISCOVER-Seq detects off-targets in cellular models and in vivo upon adenoviral gene 28 editing of mouse livers, paving the way for real-time off-target discovery during therapeutic gene 29 editing. DISCOVER-Seq is furthermore applicable to multiple types of Cas nucleases and provides 30 an unprecedented view of events that precede repair of the affected sites. 31 strengths, they also have certain weaknesses. Naïve prediction algorithms are for the most part 41 based on sequence similarity and currently have limited predictive power with very high false-42 positive rates (10). Assays that induce DSBs in vitro, such as Digenome-Seq (5), CIRCLE-Seq (6) 43 and SITE-Seq (7), have high sensitivity but dramatically under-or overestimate the number of 44 target sites that are actually modified in cellular models or in vivo (11). Nuclease concentration 45 within the cell (7), delivery method (ribonucleoprotein (RNP) vs. plasmid) (7, 12, 13) as well as 46 more complex cellular properties such as chromatin accessibility (14, 15) have been shown to 47